RESUMO
Nuclear export of the viral ribonucleoprotein (vRNP) is a critical step in the influenza A virus (IAV) life cycle and may be an effective target for the development of anti-IAV drugs. The host factor ras-related nuclear protein (RAN) is known to participate in the life cycle of several viruses, but its role in influenza virus replication remains unknown. In the present study, we aimed to determine the function of RAN in influenza virus replication using different cell lines and subtype strains. We found that RAN is essential for the nuclear export of vRNP, as it enhances the binding affinity of XPO1 toward the viral nuclear export protein NS2. Depletion of RAN constrained the vRNP complex in the nucleus and attenuated the replication of various subtypes of influenza virus. Using in silico compound screening, we identified that bepotastine could dissociate the RAN-XPO1-vRNP trimeric complex and exhibit potent antiviral activity against influenza virus both in vitro and in vivo. This study demonstrates the important role of RAN in IAV replication and suggests its potential use as an antiviral target.
Assuntos
Transporte Ativo do Núcleo Celular , Antivirais , Proteína Exportina 1 , Vírus da Influenza A , Carioferinas , Replicação Viral , Proteína ran de Ligação ao GTP , Replicação Viral/efeitos dos fármacos , Humanos , Proteína ran de Ligação ao GTP/metabolismo , Proteína ran de Ligação ao GTP/genética , Antivirais/farmacologia , Animais , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/fisiologia , Carioferinas/metabolismo , Carioferinas/antagonistas & inibidores , Cães , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Células Madin Darby de Rim Canino , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Camundongos , Piperidinas/farmacologia , Influenza Humana/virologia , Células A549 , Nucleoproteínas/metabolismo , Nucleoproteínas/genética , Células HEK293 , Linhagem Celular , Núcleo Celular/metabolismo , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genéticaRESUMO
The RNA-splicing ligase RNA 2',3'-cyclic phosphate and 5'-OH ligase (RTCB) is a catalytic subunit of the tRNA-splicing ligase complex, which plays an essential role in catalyzing tRNA splicing and modulating the unfolded protein response. However, the function of RTCB in influenza A virus (IAV) replication has not yet been described. In this study, RTCB was revealed to be an IAV-suppressed host factor that was significantly downregulated during influenza virus infection in several transformed cell lines, as well as in primary human type II alveolar epithelial cells, and its knockout impaired the propagation of the IAV. Mechanistically, RTCB depletion led to a robust elevation in the levels of type I and type III IFNs and proinflammatory cytokines in response to IAV infection, which was confirmed by RTCB overexpression studies. Lastly, RTCB was found to compete with DDX21 for RNA helicase DDX1 binding, attenuating the DDX21-DDX1 association and thus suppressing the expression of IFN and downstream IFN-stimulated genes. Our study indicates that RTCB plays a critical role in facilitating IAV replication and reveals that the RTCB-DDX1 binding interaction is an important innate immunomodulator for the host to counteract viral infection.
Assuntos
Vírus da Influenza A , Influenza Humana , Humanos , RNA Helicases DEAD-box , Imunidade Inata , Vírus da Influenza A/fisiologia , Ligases , RNA Helicases , RNA de Transferência , Replicação ViralRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has resulted in significant global morbidity, mortality, and societal disruption. Currently, effective antiviral drugs for the treatment of SARS-CoV-2 infection are limited. Therefore, safe and effective antiviral drugs to combat COVID-19 are urgently required. In previous studies, we showed that 3-indoleacetonitrile, a plant growth hormone produced by cruciferous (Brassica) vegetables, is effective in treating influenza A virus infection. However, the molecular mechanisms underlying these effects remain unclear. Herein, we demonstrated that 3-indoleacetonitrile exhibits broad-spectrum antiviral activity and is effective against HSV-1 and VSV infections in vitro. This phenomenon prompted us to study its role in the anti-SARS-CoV-2 process. Interestingly, 3-indoleacetonitrile exhibited antiviral activity against SARS-CoV-2 in vitro. Importantly, tail vein injection of 3-indoleacetonitrile resulted in good antiviral activity in mouse models infected with WBP-1 (a mouse adaptation of the SARS-CoV-2 strain). Mechanistically, 3-indoleacetonitrile promoted the host interferon signalling pathway response and inhibited autophagic flux. Furthermore, we demonstrated that 3-indoleacetonitrile induced an increase in mitochondrial antiviral-signalling (MAVS) protein levels, which might be attributed to its inhibition of the interaction between MAVS and the selective autophagy receptor SQSTM1. Overall, our results demonstrate that 3-indoleacetonitrile is potently active against SARS-CoV-2 in vitro and in vivo, which may provide a foundation for further clinical testing for the treatment of COVID-19. In addition, considering its broad-spectrum antiviral effect, it should be explored whether it also has an effect on other viruses that threaten human health.
Assuntos
COVID-19 , Animais , Humanos , Camundongos , SARS-CoV-2 , Antivirais/farmacologia , Antivirais/uso terapêutico , Interferons/farmacologiaRESUMO
In this study, polyvinylidene fluoride (PVDF) composite films were prepared by fused deposition modeling, and the effects of ionic liquid (IL) content on the printability, crystallization behavior, and electrical properties of melted PVDF were systematically investigated. The results show that the addition of IL increased the temperature sensitivity of melted PVDF and decreased its viscosity, while IL acted as a plasticizer to lower the melting point of PVDF and improve its FDM printability. The imidazole cations in IL had electrostatic interactions with the -CF2- groups in PVDF, which promoted the transformation of the nonpolar phase to the polar phase in PVDF; thus, the addition of IL was beneficial to the increase in the polar ß phase. The PVDF with 20 wt.% IL contained the highest proportion of ß phase content (32.59%). Moreover, the increase in polar ß-phase content also increased the polarization strength of PVDF and improved its ferroelectric properties. PVDF with 10 wt.% IL had the highest residual polarization strength (16.87 µC/m2).
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The M1 of influenza A virus (IAV) is important for the virus life cycle, especially for the assembly and budding of viruses, which is a multistep process that requires host factors. Identifying novel host proteins that interact with M1 and understanding their functions in IAV replication are of great interest in antiviral drug development. In this study, we identified 19 host proteins in DF1 cells suspected to interact with the M1 protein of an H5N6 virus through immunoprecipitation (IP)/mass spectrometry. Among them, PSMD12, a 26S proteasome regulatory subunit, was shown to interact with influenza M1, acting as a positive host factor in IAV replication in avian and human cells. The data showed that PSMD12 promoted K63-linked ubiquitination of M1 at the K102 site. H5N6 and PR8 with an M1-K102 site mutant displayed a significantly weaker replication ability than the wild-type viruses. Mechanistically, PSMD12 promoted M1-M2 virus-like particle (VLP) release, and an M1-K102 mutation disrupted the formation of supernatant M1-M2 VLPs. An H5N6 M1-K102 site mutation or knockdown PSMD12 disrupted the budding release of the virus in chicken embryo fibroblast (CEF) cells, which was confirmed by transmission electron microscopy. Further study confirmed that M1-K102 site mutation significantly affected the virulence of H5N6 and PR8 viruses in mice. In conclusion, we report the novel host factor PSMD12 which affects the replication of influenza virus by mediating K63-linked ubiquitination of M1 at K102. These findings provide novel insight into the interactions between IAV and host cells, while suggesting an important target for anti-influenza virus drug research. IMPORTANCE M1 is proposed to play multiple biologically important roles in the life cycle of IAV, which relies largely on host factors. This study is the first one to identify that PSMD12 interacts with M1, mediates K63-linked ubiquitination of M1 at the K102 site, and thus positively regulates influenza virus proliferation. PSMD12 promoted M1-M2 VLP egress, and an M1-K102 mutation affected the M1-M2 VLP formation. Furthermore, we demonstrate the importance of this site to the morphology and budding of influenza viruses by obtaining mutant viruses, and the M1 ubiquitination regulator PSMD12 has a similar function to the M1 K102 mutation in regulating virus release and virus morphology. Additionally, we confirm the reduced virulence of H5N6 and PR8 (H1N1) viruses carrying the M1-K102 site mutation in mice. These findings provide novel insights into IAV interactions with host cells and suggest a valid and highly conserved candidate target for antiviral drug development.
Assuntos
Interações Hospedeiro-Patógeno , Vírus da Influenza A , Complexo de Endopeptidases do Proteassoma , Ubiquitinação , Proteínas da Matriz Viral , Replicação Viral , Animais , Antivirais , Linhagem Celular , Embrião de Galinha , Fibroblastos , Humanos , Vírus da Influenza A Subtipo H1N1/química , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A/genética , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/metabolismo , Vírus da Influenza A/patogenicidade , Camundongos , Mutação , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Virulência/genéticaRESUMO
Acute myeloid leukemia (AML) with MLL-rearrangement (MLL-r) comprises â¼10% of all AML cases and portends poor outcomes. Much remains uncovered on how MLL-r AML drives leukemia development while preventing cells from normal myeloid differentiation. Here, we identified that transcription factor MEF2D is a super-enhancer-associated, highly expressed gene in MLL-r AML. Knockout of MEF2D profoundly impaired leukemia growth, induced myeloid differentiation, and delayed oncogenic progression in vivo. Mechanistically, MEF2D loss led to robust activation of a CEBPE-centered myeloid differentiation program in AML cells. Chromatin profiling revealed that MEF2D binds to and suppresses the chromatin accessibility of CEBPE cis-regulatory regions. In human acute leukemia samples, MEF2D expression showed a strong negative correlation with the expression of CEBPE. Depletion of CEBPE partially rescued the cell growth defect and myeloid cell differentiation induced by the loss of MEF2D. Lastly, we show that MEF2D is positively regulated by HOXA9, and downregulation of MEF2D is an important mechanism for DOT1L inhibitor-induced antileukemia effects. Collectively, our findings suggest that MEF2D plays a critical role in human MLL-r AML and uncover the MEF2D-CEBPE axis as a crucial transcriptional mechanism regulating leukemia cell self-renewal and differentiation block.
Assuntos
Leucemia Mieloide Aguda , Fatores de Transcrição , Diferenciação Celular , Histona-Lisina N-Metiltransferase/genética , Humanos , Leucemia Mieloide Aguda/genética , Fatores de Transcrição MEF2/genética , Proteína de Leucina Linfoide-Mieloide/genéticaRESUMO
The influenza A virus (IAV) infection is usually restricted to the respiratory tract and only rarely enters the central nervous system (CNS) and causes neurological symptoms. However, the roles of host factors involved in IAV infection in the CNS remain largely undetermined. Therefore, we aimed to characterize the host responses to IAV infection in the brain. We isolated a strain of IAV H5N6, which is neurotoxic and highly pathogenic to mice. High-throughput RNA sequencing (RNA-seq) revealed 240 differentially expressed genes in IAV-infected brains. Among the significantly downregulated genes, we focused on the gene encoding progesterone receptor membrane component-1 (PGRMC1) and observed that IAV H5N6 infection clearly inhibited PGRMC1 in both neuroblastoma and glioma cells. Furthermore, treatment with AG205, a PGRMC1-specific inhibitor, or PGRMC1 knockout promoted H5N6 multiplication in vitro, while overexpression of PGRMC1 resulted in opposite effects. Furthermore, AG205 treatment or PGRMC1 knockout significantly inhibited the retinoic acid-inducible gene I (RIG-I)-mediated interferon beta (IFN-ß) signaling pathway and reduced the levels of several antiviral proteins (Mx1 and ISG15). In addition, PGRMC1-mediated regulation of IFN signaling relied on inhibition of the expression and ubiquitination of RIG-I. The loss of PGRMC1 leads to an increased susceptibility of mice (brain and lung) to influenza A virus infection. Conclusively, our results show for the first time that IAV H5N6 downregulates PGRMC1 expression to contribute to virus proliferation by inhibiting RIG-I-mediated IFN-ß production in the brain. These findings may offer new insights regarding the interplay between IAV and host factors that may impact IAV pathogenicity in the brain. IMPORTANCE Central nervous system (CNS) disease is one of the most common extra-respiratory tract complications of influenza A virus (IAV) infections. However, there is still little knowledge about IAV regulating host responses in brain. In this study, we identified progesterone receptor membrane component-1 (PGRMC1) as a novel host factor involved in the replication and propagation of IAV H5N6 in the host brain. We also observed that PGRMC1 antagonism was required for viral evasion from the host immune response during IAV infection via inhibition of the retinoic acid-inducible gene I (RIG-I)-mediated interferon beta (IFN-ß) signaling pathway and downstream antiviral gene expression. This study revealed a newly identified regulatory mechanism used by IAV H5N6 to ensure its life cycle in the CNS.
Assuntos
Antivirais/farmacologia , Sistema Nervoso Central/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Progesterona/metabolismo , Animais , Feminino , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A , Influenza Humana , Pulmão/virologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Infecções por Orthomyxoviridae , Receptores de Progesterona/genética , Transcriptoma , Replicação ViralRESUMO
Influenza A viruses are serious zoonotic pathogens that continuously cause pandemics in several animal hosts, including birds, pigs, and humans. Indole derivatives containing an indole core framework have been extensively studied and developed to prevent and/or treat viral infection. This study evaluated the anti-influenza activity of several indole derivatives, including 3-indoleacetonitrile, indole-3-carboxaldehyde, 3-carboxyindole, and gramine, in A549 and MDCK cells. Among these compounds, 3-indoleacetonitrile exerts profound antiviral activity against a broad spectrum of influenza A viruses, as tested in A549 cells. Importantly, in a mouse model, 3-indoleacetonitrile with a non-toxic concentration of 20 mg/kg effectively reduced the mortality and weight loss, diminished lung virus titers, and alleviated lung lesions of mice lethally challenged with A/duck/Hubei/WH18/2015 H5N6 and A/Puerto Rico/8/1934 H1N1 influenza A viruses. The antiviral properties enable the potential use of 3-indoleacetonitrile for the treatment of IAV infection.
Assuntos
Antivirais/farmacologia , Indóis/farmacologia , Indóis/uso terapêutico , Vírus da Influenza A/efeitos dos fármacos , Infecções por Orthomyxoviridae/tratamento farmacológico , Células A549 , Animais , Antivirais/uso terapêutico , Antivirais/toxicidade , Cães , Feminino , Humanos , Indóis/toxicidade , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/fisiologia , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/fisiologia , Vírus da Influenza A/fisiologia , Pulmão/patologia , Pulmão/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Sulfetos/farmacologia , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Cardiovascular disease, until now, is still the leading cause of death in the United States. Due to the limited regenerative capacity of adult hearts, the damage caused by heart injury cannot be reversed and eventually progress into heart failure. In need of cardiovascular disease treatment, many therapies aimed at either cell transplantation or cell regeneration have been proposed. Direct reprogramming of somatic cells into induced cardiomyocytes (iCMs) is considered to be a promising strategy for regenerative medicine. The induction of cardiomyocytes from non-myocytes can be achieved efficiently via ectopic expression of reprogramming factors both in vitro and in vivo in the mouse model, however, the generation of human induced cardiomyocyte-like cells (hiCMs) remains challenging. The inefficiency of hiCMs production called for the identification of the additional epigenetic memories in non-myocytes which might be damping the hiCM reprogramming. Here, we conducted an unbiased loss-of-function screening focusing on epigenetic regulators and identified enhancer of zeste homolog 2 (EZH2) as an important epigenetic barrier during hiCM reprogramming. We found that the removal of EZH2 via genetic knockdown or treatment of EZH2 selective degrader significantly increased the hiCM reprogramming efficiency and led to profound activation of cardiac genes and repression of collagen and extracellular matrix genes. Furthermore, EZH2 inhibitors targeting its catalytic activity also promotes hiCM reprogramming, suggesting that EZH2 may restrain cardiac conversion through H3K27me3-mediated gene repression. Indeed, genomic profiling of H3K27me3 revealed a subset of cardiac genes that remain repressed with high levels of H3K27me3 despite of the delivery of the reprogramming factors. Inhibition of EZH2, however, leads to reduced H3K27me3 occupancy and robust activation of these cardiac genes. Taken together, our data suggested that EZH2 inhibition facilitates the activation of cardiac genes in fibroblasts and eases the production of hiCMs.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste , Repressão Epigenética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Fibroblastos/metabolismo , Histonas/metabolismo , Humanos , Miócitos Cardíacos/metabolismo , Ativação TranscricionalRESUMO
Humans and avian species are prone to influenza viral infection, which may cause serious clinical consequences. Many studies have documented the critical role of host factors in the influenza virus life cycle based on human models, but knowledge about their roles in birds is very limited. In this study, using immunoprecipitation coupled with mass spectrometry, a total of 72 potential interacting proteins of influenza nucleoprotein (NP) were identified in DF-1 cells. Among these proteins, avian chaperonin containing TCP1 subunit 5 (CCT5) was demonstrated to interact with influenza A virus (IAV) NP directly, as well as polymerase basic protein 1 (PB1) and polymerase basic protein 2 (PB2) but not with polymerase acidic protein (PA). Further investigation showed that viral infection profoundly elevated the expression level of cellular CCT5, whose expression, in turn, promoted the nuclear export of NP, as well as viral polymerase activity, thereby facilitating the replication of IAV. The obtained results suggested an important role of avian CCT5 in supporting influenza virus replication, which may serve as an anti-influenza target.
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Aberrant HOXA9 expression is a hallmark of most aggressive acute leukemias, notably those with KMT2A (MLL) gene rearrangements. HOXA9 overexpression not only predicts poor diagnosis and outcome but also plays a critical role in leukemia transformation and maintenance. However, our current understanding of HOXA9 regulation in leukemia is limited, hindering development of therapeutic strategies. Here, we generated the HOXA9-mCherry knock-in reporter cell lines to dissect HOXA9 regulation. By utilizing the reporter and CRISPR/Cas9 screens, we identified transcription factors controlling HOXA9 expression, including a novel regulator, USF2, whose depletion significantly down-regulated HOXA9 expression and impaired MLLr leukemia cell proliferation. Ectopic expression of Hoxa9 rescued impaired leukemia cell proliferation upon USF2 loss. Cut and Run analysis revealed the direct occupancy of USF2 at HOXA9 promoter in MLLr leukemia cells. Collectively, the HOXA9 reporter facilitated the functional interrogation of the HOXA9 regulome and has advanced our understanding of the molecular regulation network in HOXA9-driven leukemia.
Assuntos
Sistemas CRISPR-Cas , Regulação Leucêmica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Leucemia/metabolismo , Fatores Estimuladores Upstream/metabolismo , Alelos , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Epigênese Genética , Genes Reporter , Histona-Lisina N-Metiltransferase/genética , Humanos , Proteína de Leucina Linfoide-Mieloide/genéticaRESUMO
Influenza virus cross-species transmission is restricted by the host, but viruses overcome this restriction by accumulating mutations which allow them to adapt to a new host. Among the many factors which facilitate virus host adaptation, polymerase basic protein 2 (PB2) 627 plays an important role, although the underlying molecular mechanism has not been fully understood. In a previous study, we found that histone H1.2 (encoded by HIST1H1C) regulates human or avian influenza virus replication in different ways, indicating that it might be involved in virus host adaptation. Herein, we found that HIST1H1C expression, phosphorylation and methylation levels are decreased when infected with H1N1 influenza virus and increased when infected with H5N1 influenza virus. Overexpressing the eight gene segments of the influenza virus, we found that only PB2 significantly affects HIST1H1C expression and modifications. Since the 627 site is different between the H5N1 and H1N1 influenza viruses we constructed PB2-627E (avian variant) and PB2-627K (human variant) mutant viruses, and observed that the effects of the wild-type and the mutant viruses on HIST1H1C expression and modifications are the opposite of one another. Further analysis showed that influenza virus PB2-627 regulates HIST1H1C expression via Sp1, and specifically that PB2-627K down-regulates Sp1 and HIST1H1C while PB2-627E up-regulates Sp1 and HIST1H1C. In addition, HIST1H1C can feedback regulate DNA-dependent protein kinase and euchromatic histone-lysine N-methyltransferase 1/2, leading to altered HIST1H1C phosphorylation and methylation levels, and affecting influenza virus replication accordingly. In summary, this study illustrates the mechanism of PB2-627E/K-mediated regulation of influenza virus host adaptation.
Assuntos
Adaptação Fisiológica , Interações Hospedeiro-Patógeno , Vírus da Influenza A/genética , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética , Células A549 , Células HEK293 , Histonas/metabolismo , Humanos , Vírus da Influenza A/metabolismo , Mutação , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Fator de Transcrição Sp1/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
The influenza A virus poses serious public health challenges worldwide. Strikingly, small noncoding microRNAs (miRNAs) that modulate gene expression are closely involved in antiviral responses, although the underlying mechanisms are essentially unknown. We now report that microRNA-340 (miR340) is downregulated following influenza A and other RNA virus infections, implying that host cells deplete miR340 as an antiviral defense mechanism. Accordingly, the inhibition or knockdown of endogenous miR340 clearly prevents the infection of cultured cells, whereas the forced expression of miR340 significantly enhances virus replication. Using next-generation sequencing, we found that miR340 attenuates cellular antiviral immunity. Moreover, mechanistic studies defined miR340 as a repressor of RIG-I and OAS2, critical factors for the establishment of an antiviral response. Collectively, these data indicate that host cells may lower their viral loads by regulating miRNA pathways, which may, in turn, provide new opportunities for treatment.
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Influenza A virus can evade host innate immune response that is involved in several viral proteins with complicated mechanisms. To date, how influenza A M2 protein modulates the host innate immunity remains unclear. Herein, we showed that M2 protein colocalized and interacted with MAVS (mitochondrial antiviral signaling protein) on mitochondria, and positively regulated MAVS-mediated innate immunity. Further studies revealed that M2 induced reactive oxygen species (ROS) production that was required for activation of macroautophagy/autophagy and enhancement of MAVS signaling pathway. Importantly, the proton channel activity of M2 protein was demonstrated to be essential for ROS production and antagonizing the autophagy pathway to control MAVS aggregation, thereby enhancing MAVS signal activity. In conclusion, our studies provided novel insights into mechanisms of M2 protein in modulating host antiviral immunity and uncovered a new mechanism into biology and pathogenicity of influenza A virus. Abbreviations: AKT/PKB: AKT serine/threonine kinase; Apo: apocynin; ATG5: autophagy related 5; BAPTA-AM: 1,2-Bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetrakis; BECN1: beclin 1; CARD: caspase recruitment domain; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CQ: chloroquine; DCF: dichlorodihyd-rofluorescein; DPI: diphenyleneiodonium; DDX58: DExD/H-box helicase 58; eGFP: enhanced green fluorescent protein; EGTA: ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid; ER: endoplasmic reticulum; hpi: hours post infection; IAV: influenza A virus; IFN: interferon; IP: immunoprecipitation; IRF3: interferon regulatory factor 3; ISRE: IFN-stimulated response elements; LIR: LC3-interacting region; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAVS: mitochondrial antiviral signaling protein; MMP: mitochondrial membrane potential; MOI, multiplicity of infection; mRFP: monomeric red fluorescent protein; MTOR: mechanistic target of rapamycin kinase; NC: negative control; NFKB/NF-κB: nuclear factor kappa B; PI3K: class I phosphoinositide 3-kinase; RLR: RIG-I-like-receptor; ROS: reactive oxygen species; SEV: sendai virus; TM: transmembrane; TMRM: tetramethylrhodamine methylester; VSV: vesicular stomatitis virus.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Imunidade Inata , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas da Matriz Viral/metabolismo , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/genética , Autofagossomos/imunologia , Autofagossomos/metabolismo , Autofagossomos/virologia , Autofagia/genética , Autofagia/imunologia , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Cálcio/imunologia , Cálcio/metabolismo , Células HEK293 , Células HeLa , Humanos , Imunidade Inata/genética , Vírus da Influenza A/imunologia , Vírus da Influenza A/metabolismo , Vírus da Influenza A/patogenicidade , Mitocôndrias/virologia , Dinâmica Mitocondrial/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas da Matriz Viral/genéticaRESUMO
Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused huge economic losses to the duck industry in China since 2010. Moreover, the infection has spread rapidly, posing a potential public health concern. In this study, iTRAQ approach was first used to quantitatively identify differentially expressed cellular proteins in DTMUV-infected BHK-21 cells which are usually employed to produce veterinary vaccines for DTMUV, as well as other flaviviruses by serial passage. We identified 192 differentially expressed cellular proteins, including 11 upregulated and eight downregulated proteins at 24 h postinfection (hpi), as well as 25 upregulated and 151 downregulated proteins at 48 hpi, of which TLR9, DDX3X, and DDX5 may play important roles in virus propagation. Further, DDX3X could inhibit DTMUV replication by modulating the IFN pathway via TBK1. In conclusion, our study is the first to analyze the protein profile of DTMUV-infected cells by quantitative proteomics. We believe that our findings provide valuable information in better understanding the host response to DTMUV infection. These findings are particularly important in the development of vaccine-based strategies.
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Patos , Infecções por Flavivirus/veterinária , Flavivirus/fisiologia , Doenças das Aves Domésticas/metabolismo , Proteômica/métodos , Animais , Células Cultivadas , Cricetinae , Infecções por Flavivirus/metabolismo , Infecções por Flavivirus/virologia , Doenças das Aves Domésticas/virologiaRESUMO
Influenza virus NS2 is well known for its role in viral ribonucleoprotein nuclear export; however, its function has not been fully understood. A recent study showed that NS2 might interact with HIST1H1C (H1C, H1.2). Histones have been found to affect influenza virus replication, such as the H2A, H2B, H3, and H4, but H1 has not been detected. Here, we found that H1C interacts with NS2 via its C-terminal in the nucleus and that H1C affects influenza virus replication. The H1N1 influenza virus replicates better in H1C knockout A549 cells compared to wild-type A549 cells, primarily because of the regulation of H1C on interferon-ß (IFN-ß). Further studies showed that the H1C phosphorylation mutant (T146A) decreases IFN-ß, while H1C methylation mutants (K34A, K187A) increases IFN-ß by releasing the nucleosome and promoting IRF3 binding to the IFN-ß promoter. Interestingly, NS2 interacts with H1C, which reduces H1C-IRF3 interaction and results in the inhibition of IFN-ß enhanced by H1C. In summary, our study reveals a novel function of H1C to regulate IFN-ß and uncovers an underlying mechanism, which suggests H1C plays a role in epigenetic regulation. Moreover, our results suggest a novel mechanism for the influenza virus to antagonize the innate immune response by NS2.
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Highly pathogenic H5N1 infections are often accompanied by excessive pro-inflammatory response, high viral titer, and apoptosis; as such, the efficient control of these infections poses a great challenge. The pathogenesis of influenza virus infection is also related to oxidative stress. However, the role of endogenic genes with antioxidant effect in the control of influenza viruses, especially H5N1 viruses, should be further investigated. In this study, the H5N1 infection in lung epithelial cells decreased Cu/Zn superoxide dismutase (SOD1) expression at mRNA and protein levels. Forced SOD1 expression significantly inhibited the H5N1-induced increase in reactive oxygen species, decreased pro-inflammatory response, prevented p65 and p38 phosphorylation, and impeded viral ribonucleoprotein nuclear export and viral replication. The SOD1 overexpression also rescued H5N1-induced cellular apoptosis and alleviated H5N1-caused mitochondrial dysfunction. Therefore, this study described the role of SOD1 in the replication of H5N1 influenza virus and emphasized the relevance of this enzyme in the control of H5N1 replication in epithelial cells. Pharmacological modulation or targeting SOD1 may open a new way to fight H5N1 influenza virus.
Assuntos
Virus da Influenza A Subtipo H5N1/fisiologia , Influenza Humana/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Replicação Viral , Morte Celular , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno , Humanos , Virus da Influenza A Subtipo H5N1/genética , Influenza Humana/genética , Influenza Humana/fisiopatologia , Influenza Humana/virologia , Pulmão/citologia , Pulmão/enzimologia , Pulmão/metabolismo , Pulmão/virologia , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
H5N1 influenza A virus (IAV) causes severe respiratory diseases and high mortality rates in animals and humans. MicroRNAs are being increasingly studied to evaluate their potential as therapeutic entities to combat viral infection. However, mechanistic studies delineating the roles of microRNAs in regulating host-H5N1 virus interactions remain scarce. Here, we performed microRNA microarray analysis using A549 human lung epithelial cells infected with a highly pathogenic avian influenza virus. The microRNA expression profile of infected cells identified a small number of microRNAs being dysregulated upon H5N1 influenza A virus infection. Of the differentially expressed microRNAs, miR-136 was up-regulated 5-fold and exhibited potent antiviral activity in vitro against H5N1 influenza A virus, as well as vesicular stomatitis virus. On the one hand, 3'-untranslated region (UTR) reporter analysis revealed a miR-136 binding site in the 3' UTR of IL-6. However, on the other hand, we subsequently determined that miR-136 meanwhile acts as an immune agonist of retinoic acid-inducible gene 1 (RIG-I), thereby causing IL-6 and IFN-ß accumulation in A549 cells. Overall, this study implicates the dual role of miRNA-136 in the regulation of host antiviral innate immunity and suggests an important role for the microRNA-activated pathway in viral infection via pattern recognition receptors.
Assuntos
RNA Helicases DEAD-box/imunologia , Imunidade Inata/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , MicroRNAs/imunologia , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/imunologia , Animais , Western Blotting , Linhagem Celular Tumoral , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , Cães , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/imunologia , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/genética , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/fisiologia , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-6/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/virologia , Células Madin Darby de Rim Canino , MicroRNAs/genética , Microscopia Confocal , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Imunológicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Replicação Viral/genética , Replicação Viral/imunologiaRESUMO
Swine influenza virus and Streptococcus suis are two important contributors to the porcine respiratory disease complex, and both have significant economic impacts. Clinically, influenza virus and Streptococcus suis co-infections in pigs are very common, which often contribute to severe pneumonia and can increase the mortality. However, the co-infection pathogenesis in pigs is unclear. In the present study, co-infection experiments were performed using swine H1N1 influenza virus and Streptococcus suis serotype 2 (SS2). The H1N1-SS2 co-infected pigs exhibited more severe clinical symptoms, serious pathological changes, and robust apoptosis of lungs at 6 days post-infection compared with separate H1N1 and SS2 infections. A comprehensive gene expression profiling using a microarray approach was performed to investigate the global host responses of swine lungs against the swine H1N1 infection, SS2 infection, co-infection, and phosphate-buffered saline control. Results showed 457, 411, and 844 differentially expressed genes in the H1N1, SS2, and H1N1-SS2 groups, respectively, compared with the control. Noticeably, genes associated with the immune, inflammatory, and apoptosis responses were highly overexpressed in the co-infected group. Pathway analysis indicated that the cytokine-cytokine receptor interactions, MAPK, toll-like receptor, complement and coagulation cascades, antigen processing and presentation, and apoptosis pathway were significantly regulated in the co-infected group. However, the genes related to these were less regulated in the separate H1N1 and SS2 infection groups. This observation suggested that a certain level of synergy was induced by H1N1 and SS2 co-infection with significantly stronger inflammatory and apoptosis responses, which may lead to more serious respiratory disease syndrome and pulmonary pathological lesion.
Assuntos
Vírus da Influenza A Subtipo H1N1/patogenicidade , Infecções por Orthomyxoviridae/patologia , Infecções Estreptocócicas/patologia , Streptococcus suis/patogenicidade , Doenças dos Suínos/patologia , Animais , Apoptose , Coinfecção/microbiologia , Coinfecção/patologia , Coinfecção/virologia , Regulação para Baixo , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Análise de Sequência com Séries de Oligonucleotídeos , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/virologia , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real , Sorogrupo , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/microbiologia , Streptococcus suis/genética , Streptococcus suis/isolamento & purificação , Suínos , Doenças dos Suínos/microbiologia , Doenças dos Suínos/virologia , Regulação para CimaRESUMO
The 2009 pandemic H1N1 influenza virus (pdm/09) is typically mildly virulent in mice. In a previous study, we identified four novel swine isolates of pdm/09 viruses that exhibited high lethality in mice. Comparing the consensus sequences of the PB2 subunit of human isolates of pdm/09 viruses with those of the four swine isolate viruses revealed one consensus mutation: T588I. In this study, we determined that 588T is an amino acid mutation conserved in pdm/09 viruses that was exceedingly rare in previous human influenza isolates. To investigate whether the PB2 with the T5581 mutation (PB2-T558I) has an effect on the increased pathogenicity, we rescued a variant containing PB2-588I (Mex_PB2-588I) in the pdm/09 virus, A/Mexico/4486/2009(H1N1), referred to as Mex_WT (where WT is wild type), and characterized the variant in vitro and in vivo. The results indicated that the mutation significantly enhanced polymerase activity in mammalian cells, and the variant exhibited increased growth properties and induced significant weight loss in a mouse model compared to the wild type. We determined that the mutation exacerbated PB2 inhibition of mitochondrial antiviral signaling protein (MAVS)-mediated beta interferon (IFN-ß) expression, and PB2-588I was observed to bind to MAVS more efficiently than PB2-588T. The variant induced lower levels of host IFN-ß expression than the WT strain during infection. These findings indicate that the pdm/09 influenza virus has increased pathogenicity upon the acquisition of the PB2-T588I mutation and highlight the need for the continued surveillance of the genetic variation of molecular markers in influenza viruses because of their potential effects on pathogenicity and threats to human health.